Host dna as a biomarker of crohn&#39;s disease

ABSTRACT

The invention relates to methods of analysing a sample from a subject having or suspected of having Crohn&#39;s disease for the abundance of the subject&#39;s nucleic acid (e.g., DNA) in the sample. The invention also relates to methods for measuring abundance of nucleic acid (e.g., DNA) in stool from a human subject having or suspected of having Crohn&#39;s Disease (CD). In various embodiments, an in vitro method includes analysing the relative abundance of the host (human) DNA in said sample of stool or nucleic acid extracted or isolated from a stool sample from the host (human); determining the relative abundance of the host (human&#39;s) DNA in the sample; and associating the abundance of the host (human) DNA with the host (human) providing the stool sample, or the host (human) providing the stool sample from which the nucleic acid was extracted.

RELATED APPLICATION INFORMATION

This application is continuation application of application Ser. No.15/011,314, filed Jan. 29, 2016, which claims the benefit of priority toEuropean application no. 15305142.0, filed Jan. 30, 2015, all of whichapplications are expressly incorporated herein by reference in theirentirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, is named“ENTEROM-CON-0456349_ST25.txt” and 1,669 bytes in size.

TECHNICAL FIELD OF THE INVENTION

The invention relates to a new method for diagnosing patients with Crohndisease, and/or for diagnosing the status of the disease inCrohn-suffering patients.

INTRODUCTION

Crohn's disease (CD) is a chronic inflammatory bowel disease (IBD) thatmay affect any part of the gastrointestinal tract from mouth to anus.The age of onset is generally between 15-30 years and it is equallyprevalent in women and men. The highest prevalence is found in Europeand North America with just over 300 per 100.000 persons (Molodecky etal. 2012). CD generally leads to abdominal pain, severe diarrhoea andweight disorders. The disease is of unknown aetiology andmultifactorial: environmental factors, host genetics and gut microbiomehave all been shown to impact the risk of disease and its severity (Cho,J. H., & Brant, S. R. (2011)).

The clinical diagnosis of CD is supported by serologic, radiologic,endoscopic, and histologic findings. Currently, there are no standalonelaboratory developed tests that allow the diagnostic of CD. Amongstavailable laboratory tests, serum CRP, faecal calprotectin andlactoferrin are the most widely used markers, but they are not specificto CD. Disease activity can be measured by the Crohn's Disease Activityindex (CDAI), a score resulting from the combination of multipleparameters or the Harvey-Bradshaw index (HBI) which only consists ofclinical parameters (Laas et al. 2014).

Moreover, in patients diagnosed with CD, monitoring clinical symptomsalone is not reliable enough to assess disease activity. Patientsself-reporting low disease activity often present intestinal lesionsduring an endoscopic exam. Biological markers, such as faecalcalprotectin, are useful, but nonspecific and their increase isassociated with systemic/mucosal inflammation at the late onset of theflare. Endoscopy enables to detect mucosal healing, which is consideredas the most robust and reliable sign of disease remission; however,routine repeated endoscopic monitoring is not feasible, because of therequired bowel preparation and general anaesthesia. New imaging tools,such as MRI, have been shown to be effective, but it is expensive,time-consuming, and limited access precludes routine use. The MREnterography, presented as the most promising approach, implies alsobowel preparation and invasive colonoscopy.

The tight control of CD, through accurate surveillance and treatmentadjustment, is thus a key in the management of such patients, because ofthe recurring and remitting nature of these disorders. Yet, none of thecurrent diagnostic methods is satisfactory, for the above-mentionedreasons.

Patients and healthcare providers are therefore actively looking fornon-invasive tools enabling evaluation of disease activity andmonitoring of patients care.

More precisely, there remains a need to identify a biomarker of CD thatwould allow diagnosing the disease in a patient that is non-invasive,simple and accurate manner. This is precisely the subject of the presentinvention.

Also, there is a need for identifying a biomarker which could help indistinguishing between patients suffering from an active CD vs. from aquiescent stage of said disease. Indeed, this information could helpclinicians in diagnosing the stage of CD, predicting the occurrence ofsaid changes, in order to choose from the different treatment options(intensive or conventional), without having to perform an endoscopicanalysis. This need is fulfilled by the present invention.

Increase of host DNA in stool samples of patients suffering fromdiseases known to induce an inflammatory state of the gut mucosa hasbeen observed in the prior art.

In a prospective cohort of 599 hospitalized patients, a single rectalswab was obtained from each patient within 7 days of admission to thehospital. Host DNA proportions were negatively correlated withintestinal microbiota diversity. Enterococcus and Escherichia wereenriched in patients excreting high quantities of human DNA, whileRuminococcus and Odoribacter were depleted. The quantification of humanDNA in faeces could serve as a simple and non-invasive approach toassess bowel inflammation (Vincent et al, 2014).

Stool samples from colorectal cancer patients also contain increasedconcentrations of human DNA (Klaassen et al 2003).

The amount of host DNA in stool samples of Crohn suffering patients hasnever been assessed so far. Yet, it has been observed that stool samplesfrom CD patients also contained increased concentrations of human DNA.This is very surprising since the total area of intestinal lesions islow in CD patients (as compared with ulcerative colitis) and visiblebleeding in the faeces is quite uncommon in Crohn's disease.

The present inventors analysed, by a quantitative metagenomic analysis,but also by qPCR, the human DNA abundance in a number of stool samplesthat have been collected from healthy controls and CD patients.Moreover, the host DNA abundance was assessed in stool samples obtainedfrom patients suffering from aggressive Crohn disease vs. from aquiescent stage of said disease.

In so doing, the present inventors observed that the presence andquantity of human DNA into stool samples are markers of Crohn's disease,and that patients suffering from aggressive Crohn disease vs. from aquiescent stage of said disease can be discriminated using this verysame biomarker.

To sum up, they demonstrated that the presence of host DNA in the faecesof CD patients may be used as a biomarker of CD, of its activity orseverity.

SUMMARY

Provided herein are methods of analysing a sample from a subject havingor suspected of having Crohn's disease for the abundance of thesubject's DNA in the sample. In one embodiment, a method includesproviding a biological sample from a subject (e.g., human); determiningthe relative abundance of the subject's (e.g., human's) DNA in thesample; and comparing the relative abundance of the subject's (e.g.,human's) DNA in the sample to a reference value and determining if therelative abundance of the subject's (e.g., human's) DNA in the sample ishigher than the reference value.

Also provided herein are methods for measuring abundance of DNA in stoolfrom a human having or suspected of having Crohn's Disease (CD). In oneembodiment, a method includes providing a sample of stool or nucleicacid extracted or isolated from a stool sample from the human;determining the relative abundance of the human's DNA in the sample; andassociating the abundance of the human DNA with the human providing thestool sample, or the human providing the stool sample from which thenucleic acid was extracted.

Further provided herein are methods for generating quantitative data andmethods for generating a quantitative data set for a subject (e.g.,human) that has or is at risk of having Crohn's Disease (CD).

In one embodiment, a method includes performing at least one assay todetermine the abundance of the subject's (e.g., human's) DNA in a samplefrom the subject (e.g., human) to generate a first dataset comprisingthe quantitative data, wherein the quantitative data represents therelative abundance of the subject's (e.g., human's) DNA in the sample,optionally compared to a reference value.

In another embodiment, a method includes providing a stool sample from asubject (e.g., human) or nucleic acid extracted or isolated from a stoolsample of a subject that has or is suspected of having Crohn Disease;transforming the stool sample or nucleic acid extracted or isolated fromthe stool sample into an analytical composition comprising copies of thesubject's DNA, wherein the copies comprise one or more primers orprobes; determining a relative abundance of the subject's DNA in thestool sample or nucleic acid extracted or isolated from the stoolsample; optionally determining a calprotectin level for the stool sampleor nucleic acid extracted or isolated from the stool sample; andoptionally determining a Harvey-Bradshaw index (HBI) score for thesubject, where the quantitative dataset comprises the relative abundanceof the subject's DNA; and/or optionally the calprotectin level and/orthe HBI score.

Moreover, provided herein are amplification methods of measuring theabundance of a target nucleic acid sequence in a sample. In oneembodiment, a method includes:

contacting a sample from a subject having or suspected of having Crohn'sDisease (CD) comprising a target nucleic acid sequence with a set ofoligonucleotide primers, each primer comprising a 5′ end and a 3′ end,wherein a first primer comprises a sequence complementary to a firstregion in the target nucleic acid sequence, and a second primercomprises a sequence complementary to a second region in the targetnucleic acid sequence, wherein the second region is downstream of thefirst region;further contacting the sample with deoxynucleotide triphosphates(dNTPs), thereby forming a mixture;allowing the first and second oligonucleotide primers to anneal to thetarget nucleic acid sequence of a) thereby forming an oligonucleotideprimer annealed target nucleic acid;exposing the oligonucleotide primer annealed target nucleic acid of stepc) to a template-dependent polymerizing agent having 5′ to 3′polymerization activity under conditions permissive for amplification,and whereby the oligonucleotide primers are extended to produceextension products; and detecting the extension products, therebymeasuring the abundance of the target nucleic acid sequence in thesample.

Additionally, provided herein are reaction solutions, such solutionsoptionally contained in a vessel. In one embodiment, a reaction solutionincludes: a polymerizing agent that synthesizes or amplifies nucleicacid; a sample comprising stool or DNA extracted or isolated from stoolof a subject (e.g., human) having or suspected of having Crohn's Disease(CD); oligonucleotide primers or oligonucleotide probe designed tospecifically hybridize to a target sequence of the subject's DNA;deoxynucleotide triphosphates (dNTPs); and

buffers or other agents permissive for detection, replication oramplification of the target sequence. Such reaction solutions may be apart of the methods and compositions set forth herein, for example, foramplification of a target sequence, for example, for analysing a samplefor the abundance of the subject's (e.g., human's) DNA in the sample.

In various aspects of the invention methods herein, performance of anassay or analysis includes: providing or obtaining a sample (e.g.,stool) from a subject such as a human, where the sample includes thesubject's DNA; contacting the sample with a detectable reagent;generating a distinct complex between the detectable reagent and thesubject's DNA; and detecting the complex to generate the quantitativedata.

Still further provided are embodiments distinct from or aspects inaddition to the invention methods, compositions, kits and reactionsolutions set forth herein. Such embodiments and aspects can be usedalone or in any combination with each other.

In particular, for example, a sample may be stool, or nucleic acidextracted or isolated from stool.

In particular, for example, the abundance of the subject's DNA isdetermined by quantitation of a genomic DNA sequence.

In particular, for example, the abundance of the subject's DNA isdetermined by quantitation of a non-transcribed region or locus ofgenomic DNA.

In particular, for example, the abundance of the subject's DNA isdetermined by quantitation of a single copy per haploid genomic DNAsequence.

In particular, for example, abundance of the subject's DNA is determinedby quantitative polymerase chain reaction (qPCR).

In particular, for example, classifying the subject or human, based uponthe relative abundance of the DNA in the sample being higher or lowerthan the reference value.

In particular, for example, the subject or human is classified if therelative abundance of the DNA in the sample is higher than the referencevalue.

In particular, for example, classifying the subject or human, if therelative abundance of the DNA in the sample is higher than the referencevalue and the amount of calprotectin in the sample is higher a secondreference value.

In particular, for example, determining the amount of calprotectin inthe sample (e.g., stool, or nucleic acid extracted or isolated fromstool).

In particular, for example, a sample that is not from a subject that hascolon cancer or a bacterial infection; and/or a sample that is not froma subject that has a clostridium difficile infection.

In particular, for example, the method is not for diagnosis of coloncancer or a bacterial infection; and/or not for diagnosis of aclostridium difficile infection.

In particular, for example, the subject has symptoms associated withCrohn's Disease (CD). In particular aspects, representative non-limitingCD symptoms include: mucosal inflammation, mucosal ulcerations, orenhanced level of inflammation markers such as platelet count, meanplatelet volume, erythrocyte sedimentation rate (ESR), serumthrombopoietin, serum erythropoietin, C-reactive protein, orosomucoid(α₁-acid glycoprotein), TNFα, Interleukins (e.g., IL1, IL2, IL6, IL8,IL10, IL15) lactoferrin or calprotectin.

In particular, for example, the subject has been diagnosed with Crohn'sDisease (CD).

In particular, for example, the subject has been or is currently beingtreated for Crohn's Disease (CD).

In particular, for example, the subject has Crohn's Disease in anunstable state.

In particular, for example, the subject has Crohn's Disease in a stablestate.

Additional embodiments and aspects of the invention are set forth below,which are appropriate alone or in combination with the above embodimentsand aspects.

DESCRIPTION OF DRAWINGS

FIG. 1 discloses the Boxplots of the percent human DNA found in stoolsamples from healthy and NASH controls (on left) and Crohn Diseasepatients (on the right).

FIG. 2 discloses the relative abundance levels (in %) of the Host DNAfound in the studied stool samples, both for Crohn disease patients inan active phase of the disease (on the left) and in a quiescent phase(on the right). The status of the disease (active phase vs quiescentphase) were determined using the combined score of calprotectin leveland HBI score.

FIG. 3 discloses the percentage of human DNA versus calprotectin levelsfound in the studied stool samples.

FIG. 4 discloses ROC curve using the percent of human DNA as apredictive score of disease activity within a CD population. The AUC hasa value of 0.67 for the combined score.

FIG. 5 discloses the correlation between the quantitative data of theVP5 and VP9 qPCR assays for measuring host DNA abundance.

FIG. 6 discloses the correlation between the quantitative data of theVP5 qPCR assay and the percentage of human DNA relative abundancemeasured by Illumina sequencing.

FIG. 7 discloses the correlation between the quantitative data of theVP9 qPCR assay and the percentage of human DNA relative abundancemeasured by Illumina sequencing.

FIG. 8 discloses the correlation between mRNA level of faecalcalprotectin by qPCR assay (measure of S100A8 and S100A9 mRNA level) andprotein level of faecal calprotectin by ELISA (A: S100A8; B: S100A9).

DETAILED DESCRIPTION Methods of Measures, in Particular to Diagnose CD

In a first aspect, the present invention relates to a method forgenerating quantitative data for a subject having or suspected of havingCrohn disease (CD), said method comprising: performing at least oneassay to determine host DNA relative abundance in a biological samplefrom said subject, wherein the quantitative data represents host DNArelative abundance preferably compared to a reference value.

In other words, the invention relates to an in vitro method foranalysing a biological sample from a subject having or suspected ofhaving Crohn disease (CD), said method comprising:

a) Obtaining a biological sample from said subject,b) Determining the relative abundance of host DNA in said sample,and,c) Determining if said relative abundance is higher than a referencevalue.

More precisely, the present invention relates to an in vitro method fordiagnosing Crohn disease (CD) in a subject, said method comprising:

a) Obtaining a biological sample from said subject,b) Determining the relative abundance of host DNA in said sample,and,c) Diagnosing that said subject suffers from Crohn disease, if saidrelative abundance is higher than a reference value.

This method is advantageous over the prior art diagnosis method as it isnon-invasive, economically acceptable, and present a high specificity.

In some embodiments a method comprises generating a quantitative datasetfor a subject, for example, by analysing or performing an assay from oneor more samples obtained from the subject. A quantitative dataset can bea combined dataset (e.g., a collection of data) comprising one or moredata obtained from one or more samples obtained from the same subject.In certain embodiments, a quantitative dataset comprises quantitativedata obtained from a sample obtained from stool.

As used herein, the term “host DNA” refers to the DNA of the host of thegut microbiota, as opposed to microbial or viral DNA. If the testedsubject is a human patient, then the term “host DNA” refers specificallyto “human DNA”.

DNA can be extracted from said biological sample of interest for exampleby using the extraction protocol described in Godon J J. et al., 1997.Other protocols can nevertheless be used and are well-known. Of note,the microbial DNA and the host DNA do not need to be physicallyseparated for subsequent analysis.

Mammal DNA can be distinguished from microbial DNA by any conventionalmean, such as detection of CpG methylation or of the bacterial 16Sribosomal DNA. It is also possible to use qPCR targeting the ALU (STR)repeat regions in human DNA, or the Beta-globulin, Beta-actin, and hTERTgenes (Klaassen C H W et al, 2003; Shewale J G et al, Journal ofForensic Science, 2007, vol. 52(2)). Nanostring technologies could bealso useful.

Quantification of the host and microbial DNA can be performed by anywell-known method. The most commonly used methods known in the art forthe quantification of DNA strands in a sample include Northern blottingand in situ hybridization (Parker & Barnes, Methods in Molecular Biology106:247-283 (1999)) PCR-based methods, such as quantitative polymerasechain reaction (qPCR) (Heid et al., Genome Research 6:986-994 (1996)),and nucleic-acid based multiplex techniques, such as multiplex PCR andDNA microoarrays. Alternatively, antibodies may be employed that canrecognize sequence-specific duplexes, including DNA duplexes orDNA-protein duplexes. Representative methods for sequencing-basedanalysis include chain-termination methods, shotgun sequencing methods,de novo sequencing, next generation sequencing methods (includingMassively Parallel Signature Sequencing (MPSS), Polony sequencing, 454pyrosequencing, Illumina (Solexa) sequencing, SOLiD sequencing, Ionsemiconductor sequencing, DNA nanoball sequencing, Helioscope singlemolecule sequencing, Single molecule real time (SMRT) sequencing, RNAPsequencing, Nanopore DNA sequencing, Sequencing by hybridization andMicrofluidic Sanger sequencing).

As used herein, a polymerizing agent having 5′ to 3′ polymerizationactivity is typically a protein that can catalyse/synthesize polymers ofnucleosides or nucleotides. Such synthesis may require a templatenucleic acid, which results in synthesis of the sequence complementaryto the nucleic acid template. Specific non-limiting examples ofpolymerizing agents having 5′ to 3′ polymerization activity include aheat stable polymerase, such as Taq polymerase.

As shown in the examples below, it is also possible to measure host DNAfrom a pool of DNA by i) sequencing the DNA present in stool samplesusing high throughput sequencing technologies and ii) by aligning theshort reads obtained by means of these sequencing technologies to thehuman genome. In this case, “relative abundance of host DNA” can becalculated by counting the number of reads mapped to a single referencesequence from the human genome (H) and the remaining number of readsgenerated (B), and normalizing the number of reads H by the total amountof reads (H+B).

As meant herein, the term “host DNA abundance” refers to the relativeamount of host DNA as compared with the total amount of DNA present insaid sample (including in particular bacterial and fungal DNA). In thepresent application, it will therefore preferably be referred to as“relative abundance” (or “relative amount”) of host DNA.

Preferably, the host DNA abundance is measured by qPCR with humanspecific nucleic acid fragments, such as primers and/or probes.

As used herein, the term “nucleic acid”, “nucleic acid sequence”,“nucleotide”, “nucleotide sequence”, which are interchangeable, refer toa precise succession of natural nucleotides (e.g., A, T, G, C and U),corresponding to a single-stranded or double-stranded DNA such as cDNA,genomic DNA, ribosomal DNA or plasmidic DNA, and the transcriptionproduct of said DNA, such as RNA. A nucleic acid according to theinvention may be isolated and prepared by any known method including,but not limited to, any synthetic method, any recombinant method, any exvivo generation method and the like, as well as combinations thereof.

The probes and primers required or useful to carry out the qPCR on hostDNA are referred herein as “nucleic acid fragments” in the context ofthe invention.

By “nucleic acid fragment”, it is more generally meant herein a nucleicacid hybridizing to a nucleic acid of interest. For instance, suchnucleic acid fragment may be at least 10 nucleotides in length orpreferably, at least 15 nucleotides in length. They may also be at least25 or at least 50 nucleotides in length.

Nucleic acid fragments according to the invention are specific to hostDNA, and preferably to human host DNA, as they allow the discriminationof host DNA from other DNA present in the biological sample (i.e. nonhost DNA), such as fungal and/or bacterial DNA (i.e. microbial DNA). Inother words, the nucleic acid fragments of the invention will hydridizeto host DNA, but not (or essentially not) bind to a substantial part ofthe other DNA present in the biological sample (i.e. non host DNA), suchas fungal and/or bacterial DNA (i.e. microbial DNA).

In the context of the present invention, the nucleic acid fragment willpreferably hybridize to the host DNA under stringent hybridizationconditions. One example of stringent hybridization conditions is whereattempted hybridization is carried out at a temperature from about 50°C. to about 65° C., more preferably from about 55° C. to about 65° C.,using a salt solution which can be e.g. about 0.9 molar. However, theskilled person will be able to vary such conditions in order to takeinto account variables such as the nucleic acid fragment length, basecomposition, type of ions present, etc

A “primer” more specifically refers to a nucleic acid fragment thatserves as a starting point for amplification of a nucleic acid ofinterest, i.e. herein of host DNA. Examples of nucleic primers of theinvention include, but are not limited to, the primers of sequence SEQID NO:1, SEQ ID NO:2, SEQ ID NO:4 and SEQ ID NO:5. Such primers can beused in “a primer set” to amplify host DNA. Examples of primer set ofthe invention include, but are not limited to, the primer sets (SEQ IDNO:1, SEQ ID NO:2), and (SEQ ID NO:4, SEQ ID NO:5).

A “probe” more specifically refers to a nucleic acid fragment that canbe used for detection of a nucleic acid of interest, i.e. herein of hostDNA. This term encompasses various derivative forms such “fluorescentprobe”. When used in combination with a primer set as defined above,said probe can be used for quantification of a nucleic acid of interest.Examples of probes of the invention include, but are not limited to, theprobes of sequence SEQ ID NO:3, and SEQ ID NO:6. Probes may be labelledby isotopes, radiolabels, binding moieties such as biotin, haptens suchas digoxygenin, luminogenic, mass tags, phosphorescent or fluorescentmoieties, or by fluorescent dyes alone (e.g., MGB, FAM, VIC, TET, NED,TAMRA, JOE, HEX, ROX, etc) or in combination with other dyes. Theselabels provide signals detectable by fluorescence, radioactivity,colorimetry, gravimetry, X-ray diffraction or absorption, magnetism,enzymatic activity, mass spectrometry, binding affinity and the like,and facilitate the detection or quantification of the nucleic acid ofinterest

A “detectable reagent” may be incorporated into or bound to the detectedproduct, by way of, for example, covalent or non-covalent bonding (e.g.,van der Waals forces, ionic bonds, hydrophobic bonds, hydrogen bonding,etc.). In the context of the invention, primers, probes and othernucleic acids may be considered as non-limiting examples of detectablereagents. Other non-limiting examples of detectable reagents includeproteins, such as antibodies. A “complex” can be used to refer to thebinding between a detectable reagent and a target, such as host/humannucleic acid (e.g., RNA or DNA), and is typically a non-naturallyoccurring entity formed by the interaction.

An analytical composition often comprises elements, matter and/orcomponents not found in nature. In certain embodiments, an analyticalcomposition comprises matter made by the hand of man, non-limitingexamples of which include copies of nucleic acids (e.g., RNA or DNA,e.g., amplicons generated in vitro often by use of a polymerizingagent), probes, primers and/or detectable reagents, nucleotideanalogues, disproportionate quantities of nucleic acid (e.g., amountsand ratios of nucleic acid fragments (e.g., copies and/or amplicons) notfound in nature (e.g., in a cell or an organism), nucleic acid in apartially pure or purified form which form excludes other biologicalcell components (e.g., whole cells, eukaryotic or prokaryotic), proteins(e.g., histones), serum, blood, carbohydrates, bacterial cell walls,organelles, lipid, etc.).

In a preferred embodiment of the invention, host DNA abundance ismeasured by quantitative PCR (qPCR) by using at least one nucleic acidfragment selected from the group of nucleic acid fragments of sequenceSEQ ID NO:1 to SEQ ID NO:6, variants thereof and complementary sequencesthereof.

More preferably, host DNA abundance is measured by quantitative PCR(qPCR) by using the primer set (SEQ ID NO:1, SEQ ID NO:2) combined withthe probe of sequence SEQ ID NO:3, and/or by using the primer set (SEQID NO:4, SEQ ID NO:5) combined with the probe of sequence SEQ ID NO:6.

The term “complementary” means that, for example, each nucleotide of afirst nucleic acid sequence is paired with the complementary base of asecond nucleic acid sequence whose orientation is reversed.Complementary nucleotides are A and T (or A and U) or C and G.

“Variants” of a nucleic acid fragment according to the present inventioninclude, but are not limited to, nucleic acid sequences which are atleast 99% identical after alignment to said nucleic acid fragment andretain their capacity to hybridize to a nucleic acid of interest, hereinto host DNA. Examples of variants are degenerate nucleic acid fragments.

The methods of the invention can be applied to any subject, either humanor animal. Yet, in a preferred embodiment, it is applied to a humanpatient, in particular to a human that is suspected of suffering fromCD, or is in need of a confirmation of having CD. More precisely, themethod of the invention is useful for monitoring human patients showingenhanced level of inflammation markers such as platelet count, meanplatelet volume, erythrocyte sedimentation rate (ESR), serumthrombopoietin, serum erythropoietin, C-reactive protein and orosomucoid(α₁-acid glycoprotein), TNFα, Interleukins (notably IL1, IL2, IL6, IL8,IL10, IL15) as well as fecal markers of inflammation such as lactoferrinand calprotectin. Precise methods for diagnosing CD are detailed in Laaset al, 2014, which is incorporated herein by reference. More preferably,the subject is not suffering from at least one of the followingpathologies: cancer or precancer, more particularly from colon cancer,colorectal cancer or colorectal adenoma, ulcerative colitis, microscopiccolitis (such as collagenous colitis or lymphocytic colitis), ischaemiccolitis, diversion colitis, allergic colitis, Behcet's disease,colorectal polyps, celiac disease, irritable bowel syndrome (IBS), andany combination thereof.

In a preferred embodiment, the biological sample used in step a) of themethod invention is a stool sample. Indeed, such a sample may beobtained by a completely harmless collection from the patient.Preferably, said stool sample is collected and stored in appropriatebuffers that do not denature or affect the DNA contained in same (inthis aim, one can use, e.g., the RNA Later® RNA stabilization Reagent(Ambion), or the Stool DNA Stabilizer (Invitek), or a mix of EDTA andDMSO). More preferably, the samples are stored at −80° C. until DNAextraction and subsequent analysis.

As used herein, the term “reference value” refers to a specific value ordataset that can be used to identify samples that are known to be poorin host DNA or to identify samples having stable CD. In someembodiments, a reference value is obtained for example from historicalabundance data obtained for healthy subjects. In some embodiments, areference value is obtained from historical abundance data obtained fora patient or a pool of patients having been diagnosed unambiguously fora stable CD. In this example, a reference value can be obtained bymeasuring the relative abundance of host DNA in stool samples frompatients being in a stable state of CD. It can be a single cut-offvalue, such as a median or mean. It can be a single number, equallyapplicable to every sample. In a preferred embodiment, this referencevalue is a predetermined value. For healthy subjects a predeterminedvalue is of about 1%. For subjects with stable CD, a predetermined valueis of about 10%.

In principle, stool samples of healthy subjects are devoid of host DNA.Therefore, the presence of host DNA in the stool samples of a subject isa hint that said subject may suffer from a gut related disease. Thepresent invention also encompasses all methods aimed at diagnosing CD ina subject, involving the detection of the presence of host DNA in stoolsamples. In other words, any diagnostic method involving the use of hostDNA as biomarker of CD is encompassed within the present invention.

In the context of the invention, it is meant that the relative abundanceof host DNA for the tested subject is “higher than a reference value” ifit is superior, preferably 10 folds, and more preferably 20 foldssuperior to said reference value. In a preferred embodiment, it can beconcluded that the tested subject is suffering from CD if the relativeabundance of host DNA, as defined above, is higher than 1%, preferablyhigher than 10%, more preferably higher than 20%.

In other terms, the amount of host DNA is compared with a referencevalue. Said comparison can be done by those skilled in the art usingstatistical methods, in particular a ROC curve can be used to determinean optimal cut-off for sensitivity and specificity.

Comparison of host DNA abundance profile of a given subject with areference value can be performed using statistical models or machinelearning methods, whose aim is to predict a clinical response (e.g., 0Mucosal Healing, 1 Mucosal Ulceration) based on a combination of theexplanatory variables (amounts of host, e.g., human) DNA andcalprotectin). Particular statistical models such as logistic regressionand fisher linear discriminant analysis are relevant to predict outcome.Other discriminating algorithms include kNN (k nearest neighbour),decision trees, SVM (support vector machine), NN (neural networks) andforest

In a particular embodiment, the method of the invention comprises:performing at least one assay to determine host DNA relative abundancein a stool sample from a subject having or suspected of having Crohndisease (CD), wherein the quantitative data represents host DNA relativeabundance preferably compared to a reference value of about 1%.

In other words, the invention relates to an in vitro method foranalysing a biological sample from a subject having or suspected ofhaving Crohn disease (CD), said method comprising:

a) Obtaining a stool sample from said subject,b) Determining the relative abundance of host DNA in said sample,and,c) Determining if said relative abundance is higher than a referencevalue of about 1%.

More precisely, in this particular embodiment, the method of theinvention comprises the following steps:

a) Obtaining a stool sample from a human patient,b) Determining the relative abundance of human DNA in said sample, byany conventional means disclosed above, and,c) Diagnosing that said patient suffers from Crohn disease, if saidrelative abundance is higher than about 1%.

Methods of Measures, in Particular to Monitor the States of CD

In another aspect, the present invention relates to a method forgenerating quantitative data for a subject having or suspected of havingCrohn disease (CD) in an unstable state, said method comprising:performing at least one assay to determine host DNA relative abundancein a biological sample from said subject, wherein the quantitative datarepresents host DNA relative abundance preferably compared to areference value.

In other words, the invention relates to an in vitro method foranalysing a biological sample from a subject having or suspected ofhaving Crohn disease in an unstable state, said method comprising thesteps of:

a) Obtaining a biological sample from said subject,b) Determining the relative abundance of host DNA in said sample, and,c) Determining if said relative abundance is higher than a referencevalue.

In a preferred embodiment, said reference value is of about 10%.

The present invention further relates to a method for generatingquantitative data for a subject having or suspected of having Crohndisease (CD) in a stable state, said method comprising: performing atleast one assay to determine host DNA relative abundance in a biologicalsample from said subject, wherein the quantitative data represents hostDNA relative abundance preferably compared to a reference value.

In a preferred embodiment, said reference value is of about 1%.

In other words, the invention relates to an in vitro method foranalysing a biological sample from a subject having or suspected ofhaving Crohn disease in a stable state, said method comprising the stepsof:

a) Obtaining a biological sample from said subject,b) Determining the relative abundance of host DNA in said sample, and,c) Determining if said relative abundance is lower than a firstreference value and higher than a second reference value.

In a preferred embodiment, said first reference value is of about 10%,and said second reference value is of about 1%.

More particularly, the results obtained by the inventors allowed toidentify a biomarker (host DNA) allowing to distinguish between patientssuffering from an inactive (quiescent state) Crohn disease from patientssuffering from aggressive Crohn disease (state associated with animminent flare period), in particular in a non-invasive manner. Theirresults are consequently of peculiar value with regard to monitoring thestage of this disease.

In the context of the invention, “stable” patients are defined as CDpatients for whom disease activity is stable over several weeks (patientin a “stable state”). While “unstable” patients (or patient “in anunstable state”) are CD patients who had their treatment changed orintensified in the following weeks, whose blood tests showed/showselevated activity in the following weeks, and/or whose self-evaluationshowed/shows decreased health and/or had/have elevated calprotectinlevels in consecutive samples, and/or who had/have systemic mucosalinflammation, more particularly systemic mucosal ulcerations. Stable orunstable can also be defined based on colonoscopical scores such asCDEIS or SES-CD.

Accordingly, the present invention more particularly targets a methodaiming at diagnosing these two particular states in a subject sufferingfrom CD.

More precisely, the present invention relates to an in vitro method fordiagnosing the activity of the Crohn Disease in a subject, said methodcomprising the steps of:

a) Obtaining a biological sample from said subject,b) Determining the relative abundance of host DNA in said sample, and,c) Diagnosing that said subject has a Crohn Disease in an unstablestate, if said relative abundance is higher than a reference value.

The above methods are advantageous over the prior art as they arenon-invasive, economically acceptable, and present high specificity.

All the embodiments disclosed above, in particular for the diagnosticmethod of the invention, also applies to the present methods, notablyaimed at monitoring the activity of the Crohn disease.

In particular, said subject can be a human patient that is suspected ofsuffering from CD, or is in need of a confirmation of having CD or hasbeen diagnosed with CD. More precisely, the methods of the invention areuseful for monitoring human patients showing enhanced level ofinflammation markers such as platelet count, mean platelet volume,erythrocyte sedimentation rate (ESR), serum thrombopoietin, serumerythropoietin, C-reactive protein and orosomucoid (α₁-acidglycoprotein), TNFα, Interleukins (notably IL1, IL2, IL6, IL8, IL10,IL15) as well as fecal markers of inflammation such as lactoferrin andcalprotectin. Precise methods for diagnosing CD are detailed in Laas etal, 2014, which is incorporated herein by reference.

Also, the biological sample is preferably a stool sample, morepreferably handled as described above.

In a preferred embodiment, the relative abundance of host DNA isdetermined as disclosed above.

The present inventors have found that these methods of the invention arehighly sensitive and specific when the relative abundance of host DNA isdetermined and compared, directly or indirectly, to a reference value.

In a preferred embodiment, it can be concluded that the tested patientis suffering from unstable CD if the relative abundance of host DNA, asdefined above, is higher than 10%, preferably higher than 18%, morepreferably higher than 20%.

In a particular embodiment, the method of the invention comprises:performing at least one assay to determine host DNA relative abundancein a stool sample from a subject having or suspected of having Crohndisease in an unstable state, wherein the quantitative data representshost DNA relative abundance preferably compared to a reference value ofabout 10%.

In other words, the invention relates to an in vitro method foranalysing a biological sample from a subject having or suspected ofhaving Crohn disease in an unstable state, said method comprising thesteps of:

a) Obtaining a stool sample from said subject,b) Determining the relative abundance of host DNA in said sample, and,c) Determining if said relative abundance is higher than a referencevalue of about 10%.

More precisely, in this particular embodiment, the in vitro diagnosticmethod of the invention enables to diagnose an unstable state of theCrohn disease in a human patient, comprising the following steps:

a) measuring the relative abundance of human DNA in a stool sample ofsaid patient by any of the above-mentioned methods, and,b) determining that said patient suffers from an unstable CD, if saidrelative abundance is higher than 10%.

Conversely, the present invention also allows the generation ofquantitative data for a subject having or suspected of having Crohndisease (CD) in a stable state, or in other words the analysis of abiological sample from said subject, in particular for diagnosing astable CD. In this case, it will be concluded that a subject suffersfrom an stable CD if the relative abundance of host DNA measured in abiological sample of said subject is higher than the reference valueused for diagnosing CD (typically 1%), but lower than the referencevalue used for diagnosing an unstable state of the disease (typically10%).

The methods of the invention can include (or exclude) the stepsconsisting of obtaining the stool sample and extracting the nucleic acidmolecule from said sample, as defined above.

In principle, stool samples of subjects being in a quiescent (inactive)CD have a relative abundance of host DNA comprised between 0 and 10%(depending on the measurement technology for example). Yet, the presenceof an intermediate level of host DNA (typically between 1% and 10%) inthe stool samples of a subject is a hint that said subject may sufferfrom CD and that said CD is in a quiescent state. Moreover, the presenceof a high level of host DNA (typically superior to 10%) in the stoolsamples of a subject is a hint that said subject may suffer from CD andthat said CD is in an active state. The present invention thereforeencompasses all methods aimed at diagnosing the state of CD in asubject, involving the detection of the presence of host DNA in stoolsamples. In other words, any diagnostic method involving the use of hostDNA as biomarker of CD state is encompassed within the presentinvention.

Methods of Measures, in Particular to Design a Treatment

In another embodiment, the diagnostic methods of the invention can beused for adapting a treatment for a subject suffering from the Crohndisease.

In this embodiment, the methods of the invention therefore comprise theadditional step of designing a treatment for the diagnosed subject, saidtreatment being adapted to the particular state of CD which has beendiagnosed (such as by the method of the invention).

Thus, according to this aspect, the invention relates to a method fortreating a subject suffering from Crohn disease, comprising:

i) generating quantitative data for a subject having or suspected ofhaving Crohn disease (CD) in an unstable or stable state, according tothe above-mentioned method, andii) treating said subject with an appropriate treatment, saidappropriate treatment being chosen in those classically attributed bythe practitioner.

In other words, the invention relates to a method for treating subjectsuffering from Crohn disease, comprising:

i) analysing a biological sample from a subject having or suspected ofhaving Crohn disease in an unstable or stable state, according to theabove-mentioned method, andii) treating said subject with an appropriate treatment, saidappropriate treatment being chosen in those classically attributed bythe practitioner.

More preferably, the invention encompasses a method for treating asubject suffering from Crohn disease, said method comprising thefollowing steps:

i) diagnosing the activity of CD in a subject according to theabove-mentioned method, andii) treating said subject with an appropriate treatment, saidappropriate treatment being chosen in those classically attributed bythe practitioner once said state of CD is diagnosed.

For example, if a CD patient is diagnosed in an unstable state, anadapted treatment can be a pharmacological treatment chosen in the groupconsisting of: azathioprine, mesalamine, abatacept, adalimumab,anakinra, certolizumab, etanercept, golimumab, infliximab, rituximab,tocilizumab, natalizumab, corticosteroids, cyclosporine, methotrexate,tacrolimus, Anti-JAK (tofacitinib), anti-integrins (Vedolizumab, rhuMAbBeta7, MAdCAM-1 Antagonist), or Anti IL12/IL23 (Ustekinumab, ABT874).

Alternatively, if a Crohn patient is diagnosed in a stable state, anadapted treatment will be lifestyle interventions, for example diets ofdifferent caloric restriction intensities and macronutrient composition(low carbohydrate, low fat, saturated fat diets).

Moreover, it is possible to use the methods of the invention for testingthe efficiency of a treatment in a subject suffering from CD, inparticular CD in an unstable state, or to evaluate the response of apatient to a treatment.

In this embodiment, the method of the invention comprises the followingsteps:

i) generating quantitative data for a subject having or suspected ofhaving Crohn disease (CD) in an unstable state, according to theabove-mentioned method, before and after the administration of atreatment, andii) concluding that the treatment is efficient in said subject if thestate before the administration of the treatment was unstable butbecomes stable upon administration of the treatment

In other words, the method of the invention comprises:

i) analysing a biological sample from a subject having or suspected ofhaving Crohn disease in an unstable state, according to theabove-mentioned method, before and after the administration of atreatment, andii) concluding that the treatment is efficient in said subject if thestate before the administration of the treatment was unstable butbecomes stable upon administration of the treatment

More precisely, the method of the invention comprises:

i) diagnosing the activity of CD before and after the administration ofa treatment, according to the above-mentioned method, andii) concluding that the treatment is efficient in said subject if thestate before the administration of the treatment was unstable butbecomes stable upon administration of the treatment

If the Crohn patient is diagnosed to be “unstable” before theadministration of the treatment and becomes “stable” upon administrationof the treatment, then said patient is responding to said treatment.This efficient treatment should therefore be preferentially maintained.

Conversely, if the Crohn patient is diagnosed to be “unstable” beforethe administration of the treatment and remains “unstable” uponadministration of the treatment, then said patient is not responding tosaid treatment, and it is better to replace said treatment with anotherone or to combine it with another treatment

Of note, if the Crohn patient is diagnosed to be “stable” before theadministration of the treatment, then it is not worth administering anychemical treatment, as lifestyle interventions could be sufficient.

Combined Methods of Measures, in Particular for Diagnosis

The present inventors furthermore propose to associate the measure ofhost DNA abundance with the measure of another biomarker commonly usedto diagnose CD, and/or the state of CD (i.e., active vs quiescentstate).

In a particular embodiment, the present invention is therefore drawn toa method for generating quantitative data for a subject having orsuspected of having Crohn disease (CD), said method comprising:

a) performing at least one assay to determine host DNA relativeabundance in a biological sample from said subject, wherein a firstquantitative data represents host DNA relative abundance preferablycompared to a reference value; andb) performing at least one assay to determine calprotectin level, or acombined clinical score, in another biological sample from said subject,wherein a second quantitative data represents said calprotectin levelpreferably compared to 150 μg/mL or said combined clinical scorepreferably compared to a predetermined score.

In other words, the invention relates to a method for analysing abiological sample from a subject having or suspected of having Crohndisease (CD), said method comprising:

a) Obtaining a biological sample from said subject,b) Determining the relative abundance of host DNA in said sample,c) Determining the calprotectin level, or a combined clinical score, inanother biological sample from said subject, and,d) Determining if said relative abundance is higher than a referencevalue, and if said calprotectin level is greater than 150 μg/mL or ifsaid combined clinical score is higher than a predetermined score.

More precisely, the invention relates to a method for diagnosing Crohndisease (CD) in a subject comprising:

a) Obtaining a biological sample from said subject,b) Determining the relative abundance of host DNA in said sample,c) Determining the calprotectin level or a combined clinical score inanother biological sample from said subject,and,d) Diagnosing that said subject suffers from Crohn Disease, if saidrelative abundance is higher than a reference value, and if saidcalprotectin level is greater than 150 μg/mL or if said combinedclinical score is higher than a predetermined score.

In a preferred embodiment, said reference value is of about 1%.

The skilled practitioner in the art would readily understand that thecalprotectin level indicated in μg/mL (or in μg/g) refers to thecalprotectin protein level, or in other words to the calprotectinprotein expression level. Protein expression level can be assessed byany method well-known in the art, notably reviewed by Reeves et al.(2000) and Schena (2005). Those methods generally involve contacting abiological sample of interest with one or more detectable reagents thatis or are suitable for measuring protein expression level, such as anantibody, and subsequently determining protein expression level based onthe level of detected reagent, preferably after normalization. Examplesof methods which generally involve the use of an antibody include,without limitation, Western blot, immunoblot, enzyme-linkedimmunosorbant assay (ELISA), enzyme-linked immunospot (ELISPOT),radioimmunoassay (RIA), immunohistochemistry and immunoprecipitation.Other methods suitable for measuring a protein expression level, whichdo not necessarily involve the use of an antibody, may be used,including, without limitation, fluorescence activated cell sorting(FACS), microscopy such as atomic force microscopy, flow cytometry,microcytometry, protein binding assay, ligand binding assay, microarray,polyacrylamide gel electrophoresis such as SDS-PAGE, surface plasmonresonance (SPR), Forster resonance energy transfer (FRET),Bioluminescence resonance energy transfer (BRET), chemiluminescence,fluorescent polarization, phosphorescence, mass spectrometry such asliquid chromatography mass spectrometry (LC-MS) or liquidchromatography/mass spectrometry/mass spectrometry (LC-MS-MS),matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF),surface-enhanced laser desorption/ionization time-of-flight (SELDI-TOF),and magnetic resonance imaging (MRI).

In another preferred embodiment, host DNA relative abundance andcalprotectin level can be measured from the same biological sample ofthe subject

Accordingly, the present invention further relates to a method forgenerating quantitative data for a subject having or suspected of havingCrohn disease (CD), said method comprising:

-   a) performing at least one assay to determine host DNA relative    abundance in a biological sample from said subject, wherein a first    quantitative data represents host DNA relative abundance preferably    compared to a reference value; and-   b) performing at least one assay to determine calprotectin level, or    a combined clinical score, in said sample, wherein a second    quantitative data represents said calprotectin level preferably    compared to 150 μg/mL or said combined clinical score preferably    compared to a predetermined score.

In other words, the invention relates to a method for analysing abiological sample from a subject having or suspected of having Crohndisease (CD), said method comprising:

a) Obtaining a biological sample from said subject,b) Determining the relative abundance of host DNA in said sample,c) Determining the calprotectin level, or a combined clinical score, insaid sample, and,d) Determining if said relative abundance is higher than a referencevalue, and if said calprotectin level is greater than 150 μg/mL or ifsaid combined clinical score is higher than a predetermined score.

More precisely, the invention relates to a method for diagnosing Crohndisease (CD) in a subject comprising:

a) Obtaining a biological sample from said subject,b) Determining the relative abundance of host DNA in said sample,c) Determining the calprotectin level, or a combined clinical score, insaid sample, and,d) Diagnosing that said subject suffers from Crohn Disease, if saidrelative abundance is higher than a reference value, and if saidcalprotectin level is greater than 150 μg/mL or if said combinedclinical score is higher than a predetermined score.

In a preferred embodiment, said reference value is of about 1%.

In a preferred embodiment, calprotectin is measured in stool samples ofthe tested subject. In a more preferred embodiment, host DNA andcalprotectin detection as described above are performed from the samestool sample. This may nevertheless require conducting two separatetypes of detection, one for measuring host DNA relative abundance (e.g.by qPCR), and one for measuring calprotectin protein level (e.g. byELISA).

Thus, in another aspect, the present invention proposes to measure thegene level of calprotectin, so that a single type of detection can beperformed in the method of the invention. More preferably, suchdetection is performed in the same container, and even more preferablyfrom the same biological sample, such as a stool sample.

In this regard, the inventors have herein surprisingly discovered thatcalprotectin protein and gene levels correlate with one another, eventhough the behaviour of these types of functional entities (i.e. geneand protein encoded by said gene) cannot be predicted from each other.Indeed, it is well-known in the art that, once transcribed, a proteinexpression level may still be regulated at the translation level, andthat the corresponding protein can be subjected to posttranslationalmodifications, varying half-lives, and compartmentalization.

Thus, according to this aspect, the invention relates to a method forgenerating quantitative data for a subject having or suspected of havingCrohn disease (CD), said method comprising:

-   a) performing at least one assay to determine host DNA relative    abundance in a biological sample from said subject, wherein a first    quantitative data represents host DNA relative abundance preferably    compared to a first reference value; and-   b) performing at least one assay to determine calprotectin gene    level, or a combined clinical score, in said sample (preferred    herein) or in another biological sample from said subject, wherein a    second quantitative data represents said calprotectin gene level    preferably compared to a second reference value or said combined    clinical score preferably compared to a predetermined score.

In other words, the invention relates to a method for analysing abiological sample from a subject having or suspected of having Crohndisease (CD), said method comprising:

a) Obtaining a biological sample from said subject,b) Determining the relative abundance of host DNA in said sample,c) Determining the calprotectin gene level, or a combined clinicalscore, in said sample (preferred herein), or in another biologicalsample from said subject, and,d) Determining if said relative abundance is higher than a firstreference value, and if the calprotectin gene level is higher than asecond reference value or if said combined clinical score is higher thana predetermined score.

More precisely, the invention relates to a method for diagnosing Crohndisease (CD) in a subject comprising:

a) Obtaining a biological sample from said subject,b) Determining the relative abundance of host DNA in said sample,c) Determining the calprotectin gene level, or a combined clinicalscore, in said sample (preferred herein) or in another biological samplefrom said subject, and,d) Diagnosing that said subject suffers from Crohn Disease, if saidrelative abundance is higher than a first reference value, and if saidcalprotectin level is higher than a second reference value or if saidcombined clinical score is higher than a predetermined score.

In a preferred embodiment, said first reference value with regard tohost DNA is as defined above, and more preferably is of about 1%.

Gene level, or gene expression level, can be measured by any methodwell-known in the art, such as the ones described above for measuringhost and microbial DNA. Genes encoding calprotectin are well-known inthe art. In particular, human calprotectin is known to form aheterodimer made of the S100 calcium binding protein A8 (S100A8, alsoknown as calgranulin A) and the S100 calcium binding protein A9 (S100A9,also known as calgranulin B or migration inhibitory factor-relatedprotein 14 (MRP14)). The nucleotide sequence of the human S100A8 gene isavailable under the Genbank accession number: CR407674, version number:CR407674.1, while the one of the human S100A9 gene is available underthe NCBI Reference Sequence accession number: NM_002965, version number:NM_002965.3.

In a preferred embodiment, the second reference value is a specificvalue or dataset that can be used to identify samples that are known tobelong to healthy subjects (i.e. not having Crohn disease). Saidreference value can therefore be easily determined by the skilledpractitioner. It can be a single cut-off value, such as a median ormean. It can be a single number, equally applicable to every sample. Ina preferred embodiment, this reference is a predetermined value. By“higher than a second reference value”, it is thus meant herein thatcalprotectin gene level is superior than said reference value.

A particularly preferred technique for measuring host DNA relativeabundance and/or calprotectin gene level is qPCR, using for examplenucleic acid fragments (such as primers and/or probes) that are specificto the gene(s) encoding calprotectin.

By “combined clinical score”, it is herein meant any score that combinesbiological parameters with clinical parameters to produce a scorerelated to disease severity or mucosal healing in CD. It can be forexample a combination of calprotectin levels (that are typically higherthan 150 μg/mL in CD suffering patients), HBI (that is typically higherthan 4 in CD suffering patients), gender, age, disease duration,platelet count, albumin, platelet, CRP, rectal bleeding (Abstract OP05,7^(th) congress of ECCO).

By “predetermined score”, it is herein meant a value resulting from acombination of multiple parameters through any statistical oralgorithmic method (see, e.g., the parameters and values mentioned inAbstract OP05, 7th congress of ECCO). In some embodiments apredetermined score is 150m/mL for Calprotectin. In some embodiments apredetermined score is 4 for HBI.

In a further aspect, the present invention relates to a method forgenerating quantitative data for a subject having or suspected of havingCrohn disease in an unstable state, said method comprising:

-   a) performing at least one assay to determine host DNA relative    abundance in a biological sample from said subject, wherein a first    quantitative data represents host DNA relative abundance preferably    compared to a reference value; and-   b) performing at least one assay to determine calprotectin level, or    a combined clinical score, in another biological sample from said    subject, wherein a second quantitative data represents said    calprotectin level preferably compared to 250 μg/g, or said combined    clinical score preferably compared to a predetermined score.

In other words, the invention relates to an in vitro method foranalysing a biological sample from a subject having or suspected ofhaving Crohn disease in an unstable state, said method comprising thesteps of:

a) Obtaining a biological sample from said subject,b) Determining the relative abundance of host DNA in said sample,c) Determining the calprotectin level or a combined clinical score inanother biological sample from said subject, and,d) Determining if said relative abundance is greater than a referencevalue, and if said calprotectin level is greater than 250 μg/g or ifsaid combined clinical score is higher than a predetermined score.

More precisely, the present invention relates to an in vitro method fordiagnosing the activity of the Crohn Disease in a subject, comprisingthe steps of:

a) Obtaining a biological sample from said subject,b) Determining the relative abundance of host DNA in said sample,c) Determining the calprotectin level or a combined clinical score inanother biological sample from said subject, and,d) Diagnosing that said subject has a Crohn disease in an unstablestate, if said relative abundance is greater than a reference value, andif said calprotectin level is greater than 250 μg/g or if said combinedclinical score is higher than a predetermined score.

In a preferred embodiment, said reference value is of about 10%.

In another preferred embodiment, host DNA relative abundance andcalprotectin level can be measured from the same biological sample ofthe subject

Accordingly, the present invention further relates to a method forgenerating quantitative data for a subject having or suspected of havingCrohn disease in an unstable state, said method comprising:

-   a) performing at least one assay to determine host DNA relative    abundance in a biological sample from said subject, wherein a first    quantitative data represents host DNA relative abundance preferably    compared to a reference value; and-   b) performing at least one assay to determine calprotectin level, or    a combined clinical score, in said sample, wherein a second    quantitative data represents said calprotectin level preferably    compared to 250 μg/g, or said combined clinical score preferably    compared to a predetermined score.

In other words, the invention relates to a method for analysing abiological sample from a subject having or suspected of having Crohndisease in an unstable state, said method comprising the steps of:

a) Obtaining a biological sample from said subject,b) Determining the relative abundance of host DNA in said sample,c) Determining the calprotectin level or a combined clinical score, insaid sample, and,d) Determining if said relative abundance is greater than a referencevalue, and if said calprotectin level is greater than 250 μg/g, or ifsaid combined clinical score is higher than a predetermined score.

More precisely, the present invention relates to an in vitro method fordiagnosing the activity of the Crohn Disease in a subject, comprisingthe steps of:

a) Obtaining a biological sample from said subject,b) Determining the relative abundance of host DNA in said sample,c) Determining the calprotectin level or a combined clinical score, insaid sample, and,d) Diagnosing that said subject has a Crohn disease in an unstablestate, if said relative abundance is greater than a reference value, andif said calprotectin level is greater than 250 μg/g, or if said combinedclinical score is higher than a predetermined score.

In a preferred embodiment, said reference value is of about 10%.

In a preferred embodiment, calprotectin is measured in stool samples ofthe tested subject. In a more preferred embodiment, host DNA andcalprotectin detection as described above are performed from the samestool sample. This may nevertheless require conducting two separatetypes of detection, one for measuring host DNA relative abundance (e.g.by qPCR), and one for measuring calprotectin protein level (e.g. byELISA).

Thus, in another aspect, the present invention proposes to measure thegene level of calprotectin, so that a single type of detection can beperformed in the method of the invention. More preferably, suchdetection is performed in the same container, and even more preferablyfrom the same biological sample, such as a stool sample.

Thus, according to this aspect, the invention relates to a method forgenerating quantitative data for a subject having or suspected of havingCrohn disease in an unstable state, said method comprising:

-   a) performing at least one assay to determine host DNA relative    abundance in a biological sample from said subject, wherein a first    quantitative data represents host DNA relative abundance preferably    compared to a first reference value; and-   b) performing at least one assay to determine calprotectin gene    level, or a combined clinical score, in said sample (preferred    herein) or in another biological sample from said subject, wherein a    second quantitative data represents said calprotectin gene level    preferably compared to a second reference value, or said combined    clinical score preferably compared to a predetermined score.

In other words, the invention relates to a method for analysing abiological sample from a subject having or suspected of having Crohndisease (CD) in an unstable state, said method comprising:

a) Obtaining a biological sample from said subject,b) Determining the relative abundance of host DNA in said sample,c) Determining the calprotectin gene level, or a combined clinicalscore, in said sample (preferred herein) or in another biological samplefrom said subject, and,d) Determining if said relative abundance is higher than a firstreference value, and if the calprotectin gene level is higher than asecond reference value or if said combined clinical score is higher thana predetermined score.

More precisely, the invention relates to a method for diagnosing Crohndisease (CD) in an unstable state in a subject comprising:

a) Obtaining a biological sample from said subject,b) Determining the relative abundance of host DNA in said sample,c) Determining the calprotectin gene level, or a combined clinicalscore, in said sample (preferred herein) or in another biological samplefrom said subject, and,d) Diagnosing that said subject suffers from Crohn Disease in anunstable state, if said relative abundance is higher than a firstreference value, and if said calprotectin gene level is higher than asecond reference value or if said combined clinical score is higher thana predetermined score.

In a preferred embodiment, said first reference value with regard tohost DNA is as defined above, and more preferably is of about 10%; andsaid second reference value with regard to calprotectin is preferablythe calprotectin gene level observed in subjects having quiescent Crohndisease (i.e. having Crohn disease in a stable state).

In a further aspect, the present invention relates to a method forgenerating quantitative data for a subject having or suspected of havingCrohn disease in a stable state, said method comprising:

a) performing at least one assay to determine host DNA relativeabundance in a biological sample from said subject, wherein a firstquantitative data represents host DNA relative abundance preferablycompared to a reference value; andb) performing at least one assay to determine calprotectin level, or acombined clinical score, in another biological sample from said subject,wherein a second quantitative data represents said calprotectin levelpreferably compared to 250 μg/g, or said combined clinical scorepreferably compared to a predetermined score.

In other words, the invention relates to an in vitro method foranalysing a biological sample from a subject having or suspected ofhaving Crohn disease in a stable state, said method comprising the stepsof:

a) Obtaining a biological sample from said subject,b) Determining the relative abundance of host DNA in said sample,c) Determining the calprotectin level or a combined clinical score inanother biological sample from said subject, and,d) Determining if said relative abundance is greater than a referencevalue, and if said calprotectin level is lower than 250 μg/g, or if saidcombined clinical score is lower than a predetermined score.

More precisely, the invention relates to an in vitro method fordiagnosing the activity of the Crohn Disease in a subject, comprisingthe steps of:

a) Obtaining a biological sample from said subject,b) Determining the relative abundance of host DNA in said sample,c) Determining the calprotectin level or a combined clinical score inanother biological sample from said subject, and,d) Diagnosing that said subject has a Crohn disease in a stable state,if said relative abundance is greater than a reference value, and ifsaid calprotectin level is lower than 250 μg/g or if said combinedclinical score is lower than a predetermined score.

In a preferred embodiment, said reference value is of about 1%.

In another preferred embodiment, host DNA relative abundance andcalprotectin level can be measured from the same biological sample ofthe subject

Accordingly, the present invention further relates to a method forgenerating quantitative data for a subject having or suspected of havingCrohn disease in a stable state, said method comprising:

-   a) performing at least one assay to determine host DNA relative    abundance in a biological sample from said subject, wherein a first    quantitative data represents host DNA relative abundance preferably    compared to a reference value; and-   b) performing at least one assay to determine calprotectin level, or    a combined clinical score, in said sample, wherein a second    quantitative data represents said calprotectin level preferably    compared to 250 μg/g, or said combined clinical score preferably    compared to a predetermined score.

In other words, the invention relates to a method for analysing abiological sample from a subject having or suspected of having Crohndisease (CD) in a stable state, said method comprising:

a) Obtaining a biological sample from said subject,b) Determining the relative abundance of host DNA in said sample,c) Determining the calprotectin level or a combined clinical score, insaid sample, and,d) Determining if said relative abundance is greater than a referencevalue, and if said calprotectin level is lower than 250 μg/g or if saidcombined clinical score is lower than a predetermined score.

More precisely, the invention relates to a method for diagnosing Crohndisease (CD) in a stable state in a subject comprising:

a) Obtaining a biological sample from said subject,b) Determining the relative abundance of host DNA in said sample,c) Determining the calprotectin level or a combined clinical score insaid sample, and,d) Diagnosing that said subject has a Crohn disease in a stable state,if said relative abundance is greater than a reference value, and ifsaid calprotectin level is lower than 250 μg/g, or if said combinedclinical score is lower than a predetermined score.

In a preferred embodiment, said reference value is of about 1%.

In a preferred embodiment, calprotectin is measured in stool samples ofthe tested subject. In a more preferred embodiment, host DNA andcalprotectin detection as described above are performed from the samestool sample. This may nevertheless require conducting two separatetypes of detection, one for measuring host DNA relative abundance (e.g.by qPCR), and one for measuring calprotectin protein level (e.g. byELISA).

Thus, in another aspect, the present invention proposes to measure thegene level of calprotectin, so that a single type of detection can beperformed in the method of the invention. More preferably, suchdetection is performed in the same container, and even more preferablyfrom the same biological sample, such as a stool sample.

Thus, according to this aspect, the invention relates to a method forgenerating quantitative data for a subject having or suspected of havingCrohn disease in a stable state, said method comprising:

-   a) performing at least one assay to determine host DNA relative    abundance in a biological sample from said subject, wherein a first    quantitative data represents host DNA relative abundance preferably    compared to a first reference value; and-   b) performing at least one assay to determine calprotectin gene    level, or a combined clinical score, in said sample (preferred    herein) or in another biological sample from said subject, wherein a    second quantitative data represents said calprotectin level    preferably compared to a second reference value and/or to a third    reference value, or said combined clinical score preferably compared    to a predetermined score.

In other words, the invention relates to a method for analysing abiological sample from a subject having or suspected of having Crohndisease (CD) in a stable state, said method comprising:

a) Obtaining a biological sample from said subject,b) Determining the relative abundance of host DNA in said sample,c) Determining the calprotectin gene level, or a combined clinicalscore, in said sample (preferred herein), or in another biologicalsample from said subject, and,d) Determining if said relative abundance is higher than a firstreference value, and if the calprotectin gene level is lower than asecond reference value and/or higher than a third reference value or ifsaid combined clinical score is lower than a predetermined score.

More precisely, the invention relates to a method for diagnosing Crohndisease (CD) in a stable state in a subject comprising:

a) Obtaining a biological sample from said subject,b) Determining the relative abundance of host DNA in said sample,c) Determining the calprotectin gene level, or a combined clinicalscore, in said sample (preferred herein), or in another biologicalsample from said subject, and,d) Diagnosing that said subject suffers from Crohn Disease in a stablestate, if said relative abundance is higher than a first referencevalue, and if said calprotectin level is lower than a second referencevalue and/or higher than a third reference value or if said combinedclinical score is lower than a predetermined score.

In a preferred embodiment, said first reference value with regard tohost DNA is as defined above for a stable state (1%); said secondreference value with regard to calprotectin is preferably thecalprotectin gene level observed in subjects having active Crohn disease(i.e. having Crohn disease in an unstable state); and/or said thirdreference value with regard to calprotectin is preferably thecalprotectin gene level observed in healthy subjects (i.e. not havingCrohn disease).

These methods can be applied to any subject, either human or animal.Yet, in a preferred embodiment, they are applied to a human patient, inparticular to a human that is suspected of suffering from CD, or is inneed of a confirmation of having CD, or has been diagnosed for CD.

The biological sample used in the method of the invention is preferablya stool sample.

In a preferred embodiment, the relative DNA abundance is determined byusing profiling methods based on hybridization analysis ofpolynucleotides, and/or sequencing of polynucleotides described above.

As indicated above, the calprotectin level is measured according to anymethod commonly known by the one of skill in the art. Preferably,calprotectin protein level can be expressed in μg/mL, or in μg/g.

These methods have significant advantages over the prior art, inparticular compared with those involving the measure of calprotectinlevel in stool samples alone. Indeed, as known from the one of skill inthe art, such diagnostic methods are not sensitive enough, and givefalse positive results.

Moreover, it has been observed by the Inventors that the two measures(step b) and step c)) do not reflect a simple correlation: the percentof human DNA is significantly increased in the samples with calprotectinhigher than 150 μg/mL reflecting the fact that there is more human DNApresent in the stool of patients having signs of gut inflammation.Therefore, although the two measures relate they do not seem to captureexactly the same clinical characteristics of clinical disease.

A preferred technique for measuring host DNA relative abundance andcalprotectin gene level is qPCR.

Kits for Use in the Methods of the Invention

The methods described above may be performed, for example, by usingprepackaged kits, comprising or consisting of the nucleic acid fragmentsof the invention.

The invention is thus directed to a kit for use in any method of theinvention, said kit comprising, or consisting of:

-   -   a) at least one nucleic acid fragment hydridizing specifically        with host DNA;    -   and    -   b) instructions for performing said method.

As used herein, the term “instructions” refers to a publication, arecording, a diagram, or any other medium which can be used tocommunicate how to perform a method of the invention. Said instructionscan, for example, be affixed to a container which contains said kit.Preferably, the instructions for using said kit include a referencevalue.

According to a preferred embodiment, said nucleic acid fragmenthydridizing specifically with host DNA is selected from the group of thenucleic acid fragments of sequence SEQ ID NO:1 to SEQ ID NO:6, variantsthereof and complementary sequences thereof,

More preferably, said kit comprises, or consists of:

-   -   a) the primer set (SEQ ID NO:1, SEQ ID NO:2) and the probe of        sequence SEQ ID NO:3; and/or the primer set (SEQ ID NO:4, SEQ ID        NO:5) and the probe of sequence SEQ ID NO:6; and    -   b) instructions for performing said method.

Yet, even more preferably, the above kit can further comprise:

-   -   c) at least one reagent capable of specifically determining        calprotectin protein or gene level.

The term “reagent capable of specifically determining calprotectinlevel” or “reagent capable of specifically determining calprotectinexpression level” designates a reagent or a set of reagents whichspecifically recognizes calprotectin and allows for the quantificationof the expression level thereof, at the protein or gene level. Thesereagents can be for example antibodies, aptamers or affibodiesspecifically recognizing the protein calprotectin, or nucleic acidfragments such as primers and/or probes recognizing the gene(s) encodingcalprotectin. In the context of the present invention, such reagent issaid to be “specific” for calprotectin or “recognizes specifically”calprotectin if it 1) exhibits a threshold level of binding and/orhybridizing activity, and/or 2) does not significantly cross-react withtarget molecules known to be related to calprotectin. The bindingaffinity of such reagent can be easily determined by one skilled in theart, for example, by Scatchard analysis. Cross-reactivity of a reagentcan as well be easily determined by one skilled in the art, and thusneed to be further detailed herein. Examples of reagents capable ofspecifically determining the expression level of calprotectin include,without limitation, anti-calprotectin antibodies (such as the MAC387IgG1 from Invitrogen) and nucleic acid fragments hydridizingspecifically with gene(s) encoding calprotectin, such as the S100A8and/or S100A9 genes as described above.

The invention further relates to the use (in particular in vitro use) ofthe kit as described above, in any method of the invention.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described herein.

All applications, publications, patents and other references, GenBankcitations and ATCC citations cited herein are incorporated by referencein their entirety. In case of conflict, the specification, includingdefinitions, will control.

As used herein, the singular forms “a”, “and,” and “the” include pluralreferents unless the context clearly indicates otherwise. Thus, forexample, reference to “quantitative data” or a “primer” or a “probe” or“reagent” or “detectable reagent” includes a plurality of such data,primers, probes, reagents, detectable reagents, and so forth.

As used herein, numerical values are often presented in a range formatthroughout this document. The use of a range format is merely forconvenience and brevity and should not be construed as an inflexiblelimitation on the scope of the invention. Accordingly, the use of arange expressly includes all possible subranges, all individualnumerical values within that range, and all numerical values ornumerical ranges include integers within such ranges and fractions ofthe values or the integers within ranges unless the context clearlyindicates otherwise. This construction applies regardless of the breadthof the range and in all contexts throughout this patent document

The invention is generally disclosed herein using affirmative languageto describe the numerous embodiments. The invention also specificallyincludes embodiments in which particular subject matter is excluded, infull or in part, such as substances or materials, method steps andconditions, protocols, procedures, assays or analysis. Thus, even thoughthe invention is generally not expressed herein in terms of what theinvention does not include, aspects that are not expressly included inthe invention are nevertheless disclosed herein.

A number of embodiments of the invention have been described.

Nevertheless, it will be understood that various modifications may bemade without departing from the spirit and scope of the invention.Accordingly, the following examples are intended to illustrate but notlimit the scope of invention described in the claims.

EXAMPLES Example 1 1. Material and Methods 1.1. Cohort 1.1.1 CD Cohort

All participants were part of the “CrohnOmeter 1” study, which aim wasto collect stool samples from a diverse population of Crohn Diseasepatients to investigate their gut microbiome. The inclusion criteriainto the study were a clinical diagnosis of Crohn Disease and theparticipants signed an informed consent form. CrohnOmeter 1 is alongitudinal study, on average each participant provided 8 stool samplesover an 8 months period of time. A total of 99 participants wereincluded and provided stool samples. Out of the 99 participants, 68 hadtheir stool samples sequenced. In total 438 samples were sequenced.

Each study participant filled in a questionnaire each time a stoolsample was provided into the study. The questionnaire capturedinformation on the patient's health and stool characteristics. Inparticular the following information was used to evaluate the state ofdisease activity/inflammatory status:

The calprotectin level (dosed in patient stools) was measured(calprotectin is a protein marker highlighting inflammation);

The Harvey-Bradshaw index (HBI) of each patient is recorded. HBI is acomposite auto-evaluated index reflective of the general health statusof the patient. The score is based on an evaluation of generalwell-being, an evaluation of abdominal pain, the number of liquid stoolsper day, the presence of abdominal mass and the presence ofcomplications. It is widely spread for the evaluation of Crohn patientstatus.

1.1.2 Healthy Control Cohort

A control group of individuals was assembled from healthy individuals.Main exclusion criteria were the use of prescription medication andhistory of significant disease. Multiple samples were collected from thesame individuals leading to a total of 137 samples.

1.1.3 NASH Control Cohort

An additional two NASH patients from a larger study were sequenced. Theaim of the larger NASH study, called NASH2, was to collect stool samplesfrom a diverse population of NASH patients to investigate their gutmicrobiome and identify differences between NASH and simple steatosispatients. 6 samples were available from those 2 patients. This cohortalthough small, was selected as a control population for an inflammatorydisease not localized in the gut

1.2. Sample Collection and Preparation 1.2.1. Fecal Sampling

The subjects from the CD cohort were provided dedicated collection kitscontaining a DNA stabilizer and written instructions every three weeksfor the collection of a stool sample from their home. Upon collection oftwo, approx. 1 gram aliquots in a validated DNA preservation buffer(typically RNA Later®), the tubes containing the samples were shipped byregular post to the laboratory. One tube was directly stored at −80° C.as a stool suspension backup. The second tube was used for DNAextraction: three aliquots were prepared from the stool material usinghigh speed centrifugation. These three aliquots were then stored at −80°C. before DNA extraction.

The same collection kit was used for the control cohorts.

1.2.2. DNA Extraction

A frozen aliquot of each fecal sample was suspended in 2500, ofguanidine thiocyanate 0.1 M Tris (pH 7.5) and 40 μL of 10% N-lauroylsarcosine. The suspension was then submitted to vigorous bead-beating torelease DNA from microbial cells and DNA extraction was conducted usingstandard protocol (Godon et al, 1997). The DNA integrity andconcentration were evaluated by Nanodrop and Agilent and on agarose gelelectrophoresis.

1.3. Illumina Sequencing

The sequencing was performed at an ISO17025 accredited laboratory on aHiSeq 2500 Illumina sequencer. They used ISO 17025-accredited methodHSHOv4 PE100. DNA library preparation followed the manufacturer'sinstruction (Illumina). The workflow indicated by the sequencing deviceprovider was used to perform the different steps: cluster generation,template hybridization, isothermal amplification, linearization,blocking and denaturing and hybridization of the sequencing primers. Thebase-calling was performed using the provider's pipeline. The target of40 million minimum paired-end reads were generated for each sample.Sequencing read length was 100 bp.

1.4 Bioinformatics Processing

The raw reads were processed using Enterome's in house pipeline. Brieflythe pipeline is based on MOCAT (Kultima et al., 2012) and a compilationof internal scripts. It consists of quality controls, mapping andcalculation of gene abundance using MOCAT v1.3, including list ofIllumina adapters and human genome (hg19). The number of reads mappingto the human genome is based on 95% identity on 90% of the length andare returned after the quality controls steps that includes trimmingbases with a low quality score. The percent of human reads in a sampleis calculated using 1−number of reads mapping to hg19/number of readsafter trimming.

2. Results 2.1. Comparison Between Controls Versus Crohn DiseasePatients (CD)

Using the Wilcoxon rank sum test, we compared the 137 samples fromhealthy controls, 6 from NASH patients to the 438 samples from CDpatients. The p-value was highly significant for CD versus healthycontrols (p-value=1.667e−12), FIG. 1. Summary statistics for the twocohorts are provided in Table 1.

TABLE 1 Summary statistics for percent of reads mapping to the Humangenome HG19. Cohort Min. 1stQu. Median Mean 3rdQu. Max. CD 0.00002820.0003732 0.0009794 0.0318600 0.0036770 0.8986000 Healthy 1.278e−051.822e−04 3.998e−04 8.975e−04 7.883e−04 1.012e−02 NASH 0.00075240.0009039 0.0011470 0.0021420 0.0014250 0.0074630

99% of samples from healthy controls had all less than 1% human DNA intheir total stool DNA, compared to 84% of Crohn samples. Thus with acutoff value of 1%, 16% of Crohn samples could be captured. The presenceof DNA in the stool is thus highly specific.

2.2. Association to Disease Severity in Crohn Disease Patients (CD)

Since there is a highly significant difference between Crohn Diseasepatients and healthy controls in terms of percent of human DNA in thestool sample, the relation to disease severity was studied, to assesswhether the measure of host DNA relative abundance could be used as abiomarker of disease activity.

To that end, patients were classified into disease active groupsaccording to three criteria: 1) whether they had a calprotectin levelabove 150,

-   -   2) whether they had an HBI score above 4,    -   3) whether they had an HBI score above 4 or their calprotectin        level above 150 (combined score)

The difference, based on the Wilcoxon rank sum test, between percent(relative abundance) of human DNA in stool samples from patients in aquiescent (n=227) versus in stool samples of patients in an active phaseof CD (n=211) was highly significant (see FIG. 2, P-val=1.034e−09).

95.5% of the samples with more than 20% human DNA were from activepatients.

90% of the samples with more than 10% human DNA were from activepatients.

80% of the samples with more than 1% human DNA were from activepatients.

The ROC curve (FIG. 4) is a visual representation, indicating the numberof true and false positives based on various cut offs. As can be seen inthe bottom left corner, there is a very high specificity (100%, but alow sensitivity, only about 10% of patients are captured). The straightline represents a “non informative” score.

Looking at calprotectin levels on its own and comparing it to human DNA(FIG. 3), the two measures do not reflect a simple correlation however,the percent of human DNA is significantly increased in the samples withcalprotectin higher than 150 μg/ml (P-value=1.041e−07) reflecting thefact that there is more Human DNA present in the stool of patientshaving signs of gut inflammation. Interestingly, although the twomeasures relate they do not seem to capture exactly the same clinicalcharacteristics of clinical disease since, as can be seen from the FIG.3, some samples have very high calprotectin levels and no human DNA.

Example 2 1. Material and Methods 1.1. Cohort

Participants were part of the “CrohnOmeter 1” study, as described above.In total 11 stool samples were sequenced. The cohort fulfilled the samecriteria as the one of Example 1.

1.2. Sample Collection and Preparation 1.2.1. Fecal Sampling and DNAExtraction

Fecal sampling and DNA extraction were performed, according to a similarprocedure as the one detailed in Example 1 above.

1.2.2. qPCR Performed on Host DNA

The eleven samples were analyzed with the ValidPrime® assay (TATAABiosciences) and run in triplicates. ValidPrime is highly optimized andspecific to a non-transcribed locus of genomic DNA that is present inexactly one copy per haploid normal genome.

The primers were run in a final concentration of 400 nM and probe had afinal concentration of 200 nM.

A standard curve spanning 100 000 copies to 6.10 copies per reaction wasrun together with the samples, dilution factor between standards was 4×.Samples were normalized to a concentration of 4.84 ng/μl which was atleast a 10× dilution.

TABLE 2 Primers and probes for qPCR quantification of host DNAPrimers and Nucleotide probes sequence from 5′ to 3′ (SEQ ID NO:)VP5 forward AACTTGGTGCGGAGGT (SEQ ID NO: 1) VP5 reverseATCGCTTCTGATGGACAC (SEQ ID NO: 2) VP5 probe CCGCCAGACTGCAATCCATCAATGACA(SEQ ID NO: 3) VP9 forward GCGGAAACACAAGGGAA (SEQ ID NO: 4) VP9 reverseTTAGAGGCAAAAGCAAAGAA (SEQ ID NO: 5) VP9 probeACAGCTAATTAAAATTGCACAGTTCCT (SEQ ID NO: 6)

Data, Cq-Values, from CFX manager software (Bio-Rad) was generated usingthreshold method. Threshold was set to 230. Standard curves wereobtained from CFX manager software (Bio-Rad). Sample concentrations werecalculated in the CFX manager using the standard curves.

1.2.3. Statistical Analysis of qPCR Data

The percentage of human DNA estimated was calculated based on the numberof reads mapping to the human genome divided by the total number ofreads in the sample. This percentage was correlated to thequantification of human DNA using the ValidPrime assays.

The ability to predict the value of a variable (human DNA) using thevalues of another variable (qPCR assay) was typically determined from alinear regression analysis of the data, assuming there is a linearresponse between the two variables. The statistical analysis wasperformed in R.

2. Results

2.1. Quantification of Host DNA Abundance by qPCR Specific Primers

FIG. 5 demonstrates a statistically significant correlation in thequantitative data generated between the qPCR assays performed using theVP5 and VP9 primers and probes (correlation=0.964).

FIGS. 6 and 7 show a statistically significant correlation in thequantitative data generated by the qPCR assay performed using the VP5(FIG. 6) or the VP9 (FIG. 7) primers and probes, and the percentage ofhuman DNA measured by the method proposed in Example 1 (i.e. Illuminasequencing) (correlation=0.909 for VP5 assay, and 0.927 for VP9 assay).

Accordingly, the measure of host DNA abundance by qPCR, using the VP5and VP9 primers and probes described above in Table 2, can allow for thediagnosis of Crohn disease, and monitoring of the activity of saiddisease.

Example 3 1. Material and Methods 1.1. Cohort

Participants were part of the “CrohnOmeter 1” study, as described above.In total 15 stool samples were sequenced. The cohort fulfilled the samecriteria as the one of Example 1.

1.2. Sample Collection and Preparation 1.2.1. Fecal Sampling and RNAExtraction

Fecal sampling was performed, according to the procedure detailed inExample 1 above

The stool samples were then extracted with PowerMag® Microbiome RNA/DNAIsolation Kit (Cat No 27500-4-EP, MOBIO Laboratories, Inc) according tomanufacturer's instructions, with a few modifications. Briefly, 650 μllysis buffer and 100 μl phenol:chloroform:isoamyl alcohol were addeddirectly to the stool samples. As much as possible was transferred tothe glass bead plate. A homogenizer (Tissuelyser II, Qiagen) was run at30 Hz for 2×5 min. After transferring the supernatant, an extra beadbeating step was performed. The volumes added to the pellet were 300 μllysis buffer and 45 μl phenol:chloroform:isoamyl alcohol. 220 μl ofinhibitor removal solution was added to the pooled supernatant and 450μl total sample volume was further processed with KingFisher Flex(Thermo Scientific).

1.2.2. RNA Quality and Normalization

The absorbance and purity of the 15 extracted RNA/DNA samples wereanalyzed on a spectrophotometer (DropSense96, Trinean nv). The qualityof the RNA was measured in RIN-values on gel electrophoresis(BioAnalyzer, Agilent Technologies). The samples were normalized toapproximately 66.67 ng/μl based on the absorbance measurement

1.2.3. cDNA Synthesis

All samples were reverse transcribed into cDNA using TATAA GrandScriptcDNA Synthesis kit #A103 (TATAA Biocenter AB). Prior to cDNA synthesis aDNase treatment was performed using Heat&Run gDNA removal Kit (Cat No80200-50, ArticZymes) according to manufacturer's instructions. Maximumload of RNA was added to the reaction to be able to retrieve as highCq-values as possible. The reagents were mixed. Reverse transcriptionwas performed in 20 μl reaction volume on T100 (Bio-Rad Laboratories,Inc). The temperature program in table 3 was applied for the cDNAsynthesis.

1.2.4. qPCR

The 15 samples were diluted 9× after reverse transcription. qPCR wasperformed with TATAA Probe® GrandMaster Mix #TA02 (TATAA Biocenter AB)and the reagents were mixed. All samples including genomic DNA and anegative control were run in triplicates in 10 μl reactions on CFX384platform (Bio-Rad). The samples were run on using 2 genes of interest(See Table 3), ValidPrime (for genomic DNA background correction) andB2M medium and short assays (for control of physical fragmentation,large delta Cq between those two assays will indicate degradation seeBjorkman, et al. (2016). The pipetting was performed by a pipettingrobot (EpMotion 5070, Eppendorf, Germany). A 2-step temperature programwas applied and detection was performed in the FAM channel.

qPCR was performed using primers and probes designed to amplify theS100A8 and S100A9 genes, which encode protein that form the heterodimerof calprotectin. Said primers and probes can be easily designed by theskilled practitioner based on the publicly available nucleotide sequenceof these genes.

TABLE 3 Genes encoding the S100A8 and S100A9 proteins Gene SymbolProtein encoded by said gene Gene sequence S100A8 S100 calcium bindingprotein A8 Genbank CR407674.1 S100A9 S100 calcium binding protein A9NCBI RefSeq NM_002965.3

Raw data was generated using threshold method on the CFX managersoftware (Bio-Rad). qPCR data was analyzed with GenEx software (MultiDAnalyses AB) using reference gene validation with geNorm and NormFinderto evaluate the most stably expressed genes.

1.2.5. Statistical Analysis

For each gene, triplicate values were averaged. The Cq values along withthe values normalized by house-keeping genes (delta Cq values), werecompared to the log 10 transformed ELISA values obtained for measuringcalprotectin protein level, (see Example 1, value of 150 μg/mL). Thespearman correlation was computed. Additionally a cutoff of 250 μg/g wasused to classify patients as inflamed or non inflammed (Dhaliwal et al.,2015) and a Wilcoxon rank test was performed on the Cq values for eachgene to compare the two groups. The statistical analysis was performedin R.

2. Results

2.1. Quantification of Calprotectin Gene Level by qPCR Specific Primers

S100A8 and S100A9 genes had Cq values for all 12 (S100A9) to 15 (S100A8)of the tested samples. Stati

TABLE 4 Statistical data Gene Symbol Spearman Correlation P-value S100A8−0.72 0.0025 S100A9 −0.59 0.039

FIG. 8 shows a statistically significant correlation between faecal mRNAlevels in genes S100A8 and S100A9 and faecal calprotectin level (proteinlevel).

Accordingly, the measure of calprotectin gene level by qPCR, and moreparticularly of the S100A8 and/or S100A9 gene level, can allow for thediagnosis of Crohn disease, and more particularly for the monitoring ofthe activity of said disease, when preferably combined with the measureof host DNA relative abundance.

This test can further be easily performed in combination with the qPCRtest for measuring host DNA abundance described in Example 2, preferablyin a single test tube.

BIBLIOGRAPHIC REFERENCES

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1.-26. (canceled)
 27. An in vitro method for diagnosing the activity ofthe Crohn Disease (CD) in a subject suffering from CD, said methodcomprising the steps of: a) Obtaining a stool sample from said subject,b) Determining the relative abundance of host DNA in said sample, and c)Diagnosing that said subject has a Crohn disease in an unstable state,if said relative abundance is higher than a reference value.
 28. Themethod of claim 27, comprising the steps of: a) Obtaining a stool samplefrom said subject, b) Determining the relative abundance of host DNA insaid sample, c) Determining the calprotectin level, in said sample or inanother biological sample from said subject, and d) Diagnosing that saidsubject has a Crohn disease in an unstable state, if said relativeabundance is greater than a reference value, and if said calprotectinlevel is greater than 250 μg/g.
 29. The method of claim 27 or 28,wherein said reference value is about 10%.
 30. The method of claim 27,comprising the steps of: a) Obtaining a stool sample from said subject,b) Determining the relative abundance of host DNA in said sample, c)Determining the calprotectin gene level, in said sample or in anotherbiological sample from said subject, and d) Diagnosing that said subjecthas a Crohn disease in an unstable state, if said relative abundance isgreater than a first reference value, and if said calprotectin genelevel is greater than a second reference value.
 31. An in vitro methodfor testing the efficiency of a treatment in a subject suffering fromCD, or to evaluate the response of a patient to a treatment, comprisingthe following steps: a) diagnosing the activity of CD before and afterthe administration of a treatment, according to the method of claim 27,and b) concluding that the treatment is efficient in said subject if thestate before the administration of the treatment was unstable butbecomes stable upon administration of the treatment.
 32. An in vitromethod for diagnosing Crohn Disease (CD) in a subject comprising: a)Obtaining a stool sample from said subject, b) Determining the relativeabundance of host DNA in said sample, and c) Diagnosing that saidsubject suffers from Crohn disease, if said relative abundance is higherthan a reference value.
 33. The method of claim 32, wherein said humanpatient is suspected of suffering from the Crohn Disease.
 34. The methodof claim 32, wherein said reference value is of about 1%.
 35. The methodof claim 32 comprising the steps of: a) Obtaining a stool sample fromsaid subject, b) Determining the relative abundance of host DNA in saidsample, c) Determining the calprotectin level, in said sample or inanother biological sample from said subject, and d) Diagnosing that saidsubject suffers from Crohn Disease, if said relative abundance is higherthan a reference value, and if said calprotectin level is greater than150 μg/mL.
 36. (canceled)
 37. The method of claim 32, comprising thesteps of: a) Obtaining a stool sample from said subject, b) Determiningthe relative abundance of host DNA in said sample, c) Determining thecalprotectin gene level, in said sample or in another biological samplefrom said subject, and d) Diagnosing that said subject suffers fromCrohn Disease, if said relative abundance is higher than a firstreference value, and if said calprotectin gene level is higher than asecond reference value.
 38. A method for treating a subject sufferingfrom CD, comprising the steps of: a) Diagnosing the activity of CDaccording to the method of claim 27, and b) Treating said subject withan appropriate treatment.
 39. The method of claim 38, wherein, if the CDpatient is diagnosed in an unstable state, then said appropriatetreatment is chosen from the group of treatments consisting of:azathioprine, mesalamine, abatacept, adalimumab, anakinra, certolizumab,etanercept, golimumab, infliximab, rituximab, tocilizumab, natalizumab,corticosteroids, cyclosporine, methotrexate, tacrolimus, Anti-JAK(tofacitinib), anti-integrins (Vedolizumab, rhuMAb Beta7, MAdCAM-1Antagonist), or Anti IL12/IL23 (Ustekinumab, ABT874).
 40. The method ofclaim 38, wherein, if the CD patient is diagnosed in a stable state,then said appropriate treatment is a diet of different caloricrestriction intensities and macronutrient composition.
 41. The method ofclaim 38, wherein said relative abundance is the relative amount of hostDNA as compared with the total amount of DNA present in said sample. 42.The method of claim 38, wherein said abundance is measured byquantitative PCR.
 43. The method of claim 38, wherein said abundance ismeasured by using at least one nucleic acid fragment selected from thegroup of nucleic acid fragments of sequence SEQ ID NO:1 to SEQ ID NO:6,variants thereof and complementary sequences thereof.
 44. The method ofclaim 38, wherein the sample comprises stool or nucleic acid extractedor isolated from stool.
 45. The method of claim 38, wherein the sampleis not from a subject that has colon cancer or a bacterial infection ora clostridium difficile infection.
 46. The method of claim 38, whereinthe subject has symptoms associated with Crohn's Disease (CD).